Photoautotrophic growth of microalgae for omega-3 fatty acid production

ABSTRACT

The invention provides methods of cultivating microalgae photoautotrophically outdoors to prepare concentrated microalgae products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) docosahexaenoic acid, two long-chain polyunsaturated fatty acids found in fish oil that are very important for human and animal health. It also provides concentrated microalgae products containing EPA and DHA and purified lipid products containing EPA and DHA purified from microalgae.

This application claims priority under 35 U.S.C. §119 as a divisionalapplication of U.S. patent application Ser. No. 11/651,790, filed Jan.10, 2007, now U.S. Pat. No. 8,030,037, issued Oct. 4, 2011, which claimspriority under 35 U.S.C. §119 from India National Patent Application,“Photoautotrophic Growth of Microalgae for Omega-3 Fatty AcidProduction,” filed Dec. 15, 2006, Applicant Parry Nutraceuticals,Inventors Swati Sebastian Thomas and Swaminathan Kumaravel.

BACKGROUND

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are LongChain Poly-Unsaturated Fatty Acids (LCPUFA) and belong to the omega-3family. These polyunsaturated fats play a very important role in thefunction of our bodies and have been shown to be important inmaintaining brain, retina and cardiovascular health (1-9). These fattyacids also play an important role in inflammation and thus they areuseful for fighting diseases linked to inflammation, which includecardiovascular disease and arthritis (10-12).

The nutritional importance of EPA and DHA began emerging in the mid1980s. The paleolithic diet contained small and roughly equal amounts onOmega-6 and Omega-3 PUFAs (ratio of 1-2:1) (13). An imbalance of thisratio can cause many age related health problems and neurodegenerativediseases (14).

In the 1980s, the major source of these LCPUFAs in the human diet wasfrom fish or from fish oil capsules. But fish stocks are decliningthroughout the world due to overfishing. In addition, fish accumulatemethyl mercury, PCB's and other toxins in their fat tissue, and thesethus contaminate fish oils.

Fish do not synthesize EPA and DHA; they accumulate them from eatingphytoplankton or eating animals that eat phytoplankton. It is thephytoplankton and other microbes that are the primary producers of EPAand DHA. Thus, an alternative source of EPA and DHA is microorganisms,and particularly phytoplankton.

A search is on to find suitable organisms to produce EPA- andDHA-containing oils (15-19).

The main difference in fish oils and algal oils is their structure. Fishoils are storage lipids and are in the form of triacylglycerides. Thealgal lipids are a mixture of storage lipids and membrane lipids. TheEPA and DHA present in algae is mostly in the form of glycolipids and asmall percentage is in the form of phospholipids. Glycolipids are mostlypart of chloroplast membranes and phospholipids are part of cellmembranes. Since glycolipids and phospholipids comprise a maximum ofapproximately 10-15% of the dry weight of algae, EPA and DHA productionin this form is not considered economically viable. It has beensuggested that cost effective production of EPA and DHA from algae (orany other microbes) would require the use of microbial strains thatcould produce large amounts of triacyglycerides (21).

Marine algae rich in EPA or DHA are produced by hatcheries ingreenhouses or indoors in large tanks or transparent cylinders. But suchmethods are expensive and commercially not viable.

Economical ways of raising microorganisms that accumulate EPA and DHAare lacking. Microalgae have the potential to be raisedphotoautotrophically—by photosynthesis without a reduced carbon source,using CO₂ as their carbon source. Since sunlight is free and land isinexpensive in some areas, it would be advantageous to raise microalgaeoutdoors photoautotrophically with sunlight in a way that results inaccumulation of EPA and/or DHA. But culturing microalgae outdoorsphotoautotrophically is challenging because the cultures grow slowly andare prone to become contaminated when cultured outdoors. In addition,strains that may accumulate significant quantities of EPA or DHA undercarefully controlled conditions may not accumulate as much under outdoorphotoautotrophic conditions.

New sources of EPA and DHA for human nutritional supplements and asanimal feed and aquaculture feed are needed. New improved methods ofculturing microalgae to serve as a source of EPA or DHA are needed.Identification of microorganisms that are suitable sources of DHA andEPA for humans, seafood, and livestock is needed.

SUMMARY

The invention involves the discovery of successful methods of culturingmicroalgae photoautotrophically outdoors to accumulate EPA and/or DHAand thus serve as a source of EPA or DHA supplementation in humannutritional supplements or in animal feed or aquaculture feed. Oneembodiment of the invention involves the discovery that using a net overthe cultures to filter sunlight to a reduced intensity is a key tosuccessful cultivation of EPA-accumulating and DHA-accumulatingmicroalgal strains. The invention also involves diluting thephotoautotrophic cultures at a rate of about 15% to 30% per day. This issignificantly less than the maximal doubling rate of the cultures, butallows stable maintenance of the cultures in outdoor photoautotrophicconditions and good accumulation of EPA and DHA. The invention alsoinvolves harvesting cultures in the exponential phase of cell growth,instead of stationary phase, and harvesting cultures with EPA and DHApredominantly in the form of membrane lipids instead of storage lipids.Cells in stationary phase accumulate more lipid in triacylglycerides asstorage lipids. During exponential growth the cells have less storagelipid and most lipid is in membranes in the form of phospho- andglyco-diacylglycerides. The inventors have found that thephotoautotrophic cultures accumulate large amounts of EPA and DHA asphospho- and glyco-diacylglycerides when harvested in exponentialgrowth.

The invention also provides for the successful cultivation of microalgaephotoautotrophically for EPA and DHA accumulation at the relatively hightemperatures of above 20° C. or 30° C. This is important because othershave reported better EPA and DHA accumulation at low temperatures, butmicroalgae grow faster at high temperatures (15, 22). Furthermore,microalgae can best be cultured outdoors year round in the tropics, butthis requires culturing them at high temperatures unless expensiverefrigeration systems are used.

One embodiment of the invention provides a method of preparing aconcentrated microalgae composition involving: (a) cultivatingmicroalgae photoautotrophically outdoors in open ponds under filteredsunlight in continuous or batch mode at a dilution rate of less than 35%per day; (b) harvesting the microalgae in exponential phase when cellnumber is increasing at a rate of at least 20% of maximal rate; and (c)concentrating the microalgae; wherein at least 40% of lipids in themicroalgae are in the form of glycodiacylglycerides,phosphodiacylglycerides, or a combination thereof and at least 5%(preferably at least 10%) by weight of fatty acids are DHA, EPA, or acombination thereof.

Another embodiment provides a concentrated microalgae compositionprepared by a process involving: (a) cultivating microalgaephotoautotrophically outdoors in open ponds under filtered sunlight incontinuous or batch mode at a dilution rate of less than 35% per day;(b) harvesting the microalgae in exponential phase when cell number isincreasing at a rate of at least 20% of maximal rate; and (c)concentrating the microalgae; wherein at least 40% by weight of lipidsin the microalgae are in the form of glycodiacylglycerides,phosphodiacylglycerides, or a combination thereof and at least 5%(preferably at least 10%) by weight of fatty acids are DHA, EPA, or acombination thereof.

Another embodiment provides a concentrated microalgae compositionprepared by a process comprising: (a) cultivating microalgaephotoautotrophically outdoors in open ponds under filtered sunlight; and(b) concentrating the microalgae; wherein at least 20% by weight of thefatty acids in the microalgae are eicosapentaenoic acid (EPA).

Another embodiment provides a concentrated microalgae compositionprepared by a process comprising: (a) cultivating microalgaephotoautotrophically outdoors in open ponds; and

(b) concentrating the microalgae; wherein the microalgae are cultivatedat above 20° C. and the microalgae are Chlorophyta and theeicosapentaenoic acid (EPA) yield in the microalgae is at least 10mg/liter culture.

Another embodiment provides a concentrated microalgae compositionprepared by a process comprising: (a) cultivating microalgaephotoautotrophically outdoors in open ponds; and

(b) concentrating the microalgae; wherein the microalgae areThalassiosira sp. or Chaetoceros sp., and at least 20% by weight of thefatty acids in the microalgae are eicosapentaenoic acid (EPA).Preferably the EPA yield is at least 10 mg/liter culture.

Another embodiment of the invention provides a concentrated microalgaecomposition prepared by a process comprising: (a) cultivating microalgaephotoautotrophically outdoors in open ponds under filtered sunlight; and(b) concentrating the microalgae; wherein the microalgae arePrymnesiophyta and the DHA yield is at least 3 mg/liter culture, and DHAis at least 10% by weight of total fatty acids.

Another embodiment of the invention provides a food grade dietarysupplement for human consumption comprising a concentrated microalgaecomposition of the invention.

Another embodiment of the invention provides an aquaculture or animalfeed comprising a concentrated microalgae composition of the invention.

Another embodiment of the invention provides a purified lipidcomposition prepared by a process involving purifying lipids from aconcentrated microalgae composition of the invention, wherein thepurified lipids comprise at least 5% (preferably at least 10%) by weightEPA or DHA or a combination thereof.

DETAILED DESCRIPTION Definitions

The term “microalgae” as used herein refers to photosynthetic organismsthat are native to aquatic or marine habitats and are too small to beseen easily as individual organisms with the naked eye.

The term “photoautotrophic” as used herein refers to growth with lightas the primary source of energy and carbon dioxide as the primary sourceof carbon.

As used herein, the term “a dilution rate of [e.g.] 30% per day” meansthat 30 ml of medium is added to 100 ml of culture each day, eithercontinuously over the course of a day or in a single batch addition eachday. The term “a dilution rate of less than X % per day” means that theaverage dilution rate over a period of days is less than X % per day andthat no individual dilution during culturing is greater than X % in asingle day.

As used herein, the term “maximal rate” of cell number increase refersto the maximal rate achieved at any stage during the outdoorphotoautotrophic growth of the particular harvested culture beingreferenced.

As used herein, cultivating microalgae “outdoors in open ponds” meanscultivating them exposed to unfiltered outdoor air. The ponds may becovered with a fabric cover that shades the pond or filters sunlightprovided the pond is exposed to unfiltered outdoor air.

Description

The invention provides various methods for culturing microalgaephotoautotrophically outdoors to produce EPA and DHA. One method used isfiltering sunlight to reduce the light intensity on the photoautotrophicculture. Shade cloth or netting can be used for this purpose. We havefound for most strains that the optimal solar intensity for growth,maintaining a pure culture, and omega-3 fatty acid accumulation is about40,000 to 50,000 lux, approximately half of the 110,000 lux of fullsunlight. Shade cloth or netting is suitable for filtering the sunlightto the desired intensity.

Another method used to successfully culture microalgaephotoautotrophically outdoors to produce EPA and DHA is to use smalldilutions and a slow dilution rate of less than 40% per day, preferablyless than 35% per day, more preferably from about 15% to about 30% perday. In other embodiments, the dilution rate is 15-40% per day or 15-35%per day. In other embodiments, the dilution rate is 10-30%, 10-35%, or10-40% per day. These smaller dilutions and lower dilution rates thanare typically used help prevent contamination in outdoorphotoautotrophic cultures. It also promotes thick culture growth thatgives good DHA or EPA yield.

Another method used to successfully culture microalgaephotoautotrophically outdoors to produce EPA and DHA is to harvest themicroalgae in exponential phase rather than stationary phase. Harvestingin exponential phase reduces the risk of contamination in outdoorphotoautotrophic cultures and has surprisingly been found to give goodyield of EPA and DHA. Typically, to drive fat accumulation in microbialcultures, the cultures are harvested in stationary phase, since cells instationary phase tend to accumulate storage lipids. But the inventorshave found that EPA and DHA accumulate to large amounts as membranelipids in cultures harvested in the exponential phase. The membranelipids containing EPA and DHA are predominantly phosphodiacylglyceridesand glycodiacylglycerides, rather than the triaclyglycerides found instorage lipids. The cultures are typically harvested when cell number isincreasing at a rate at least 20% of the maximal rate, i.e., the maximalrate achieved at any stage during the outdoor photoautotrophic growth ofthe harvested culture. In specific embodiments, the cultures areharvested in exponential phase when cell number is increasing at a rateof at least 30%, at least 40%, or at least 50% of maximal rate.

With these techniques and the strains grown, the inventors have alsoachieved good DHA and EPA yields with culture outdoors at hightemperatures. Others have reported that DHA and EPA accumulate better atlow temperatures, and that a cold shock step, where a culture is grownat higher temperatures but then shifted to low temperatures, e.g., 12°C., for several days before harvest, is needed to accumulate omega-3fatty acids (15, 22). The inventors have found that DHA and EPAaccumulate to good levels in the strains used even with growth at 30-35°C. in the Indian summer. Thus, some embodiments of the invention involveoutdoor photoautotrophic growth at at least 20° C., at least 25° C., orat least 30° C. As used herein, reference to growth at at least a giventemperature means that the culture is maintained at at least thattemperature for a majority of the outdoor photoautotrophic culture timeand a majority of the last 72 hours, 48 hours, and 24 hours before theculture is harvested.

Preferably the cultures are maintained outdoors photoautotrophically forat least 14 days.

In preferred embodiments of the methods and compositions of theinvention, the microalgae are not genetically modified by recombinantDNA techniques.

In some embodiments of the methods and compositions of the invention,the microalgae are diatoms. In some embodiments the diatoms areThalassiosira sp. or Chaetoceros sp.

In some embodiments, particularly with diatoms, or with Thalassiosirasp. or Chaetoceros sp., at least 20% by weight of the fatty acids in themicroalgae are EPA.

In other embodiments, the microalgae are Chlorophyta (green algae). Insome embodiments, the Chlorophyta are Tetraselmis sp.

In some embodiments, particularly where the microalgae are Chlorophyta,the EPA yield in the microalgae is at least 10 mg/liter culture.

Microalgae suitable for DHA production include Prymnesiophyta, morespecifically those of class Prymnesiophyceae, more specifically those oforder Isochrysales, more specifically Isochrysis sp. or Pavlova sp.

In some embodiments of the invention, the DHA yield is at least 3mg/liter culture and DHA is at least 10% by weight of fatty acids in themicroalgae. In other embodiments, the DH yield is at least 5 mg/literculture and DHA is at least 10% by weight of fatty acids in themicroalgae.

In particular embodiments, CO₂ is added to the open ponds duringcultivation. This helps to neutralize pH and to enhance photoautotrophicgrowth.

In the methods of the invention, the EPA and DHA are typicallypredominantly membrane lipids and phosphodiacylglycerides orglycodiacylglycerides. In some embodiments, at least 40% of the EPA orDHA are in the form of phosphodiacylglycerides or glycodiacylglyceridesor a combination thereof. In some embodiments, at least 30%, at least50%, or at least 60% of the EPA or DHA in the microalgae are in the formof phosphodiacylglycerides or glycodiacylglycerides or a combinationthereof.

The invention also provides a purified lipid composition prepared by aprocess comprising: purifying lipids from any of the concentratedmicroalgae compositions of the invention, wherein the purified lipidscomprise at least 5% by weight EPA or DHA or a combination thereof. Inparticular embodiments, at least 20% by weight of fatty acids in thelipid composition are EPA. In other embodiments, at least 10% by weightof the fatty acids in the lipid composition are DHA.

In some embodiments of the purified lipid compositions, at least 30%, atleast 40%, at least 50%, or at least 60% of the EPA or DHA or both inthe composition are in phosphodiacylglycerides or glycodiacylglyceridesor a combination thereof.

The invention also provides a food grade dietary supplement for humanconsumption containing a concentrated microalgae composition of theinvention or a purified lipid composition of the invention.

Another embodiment of the invention provides an aquaculture feed oranimal feed containing a concentrated microalgae composition of theinvention or a purified lipid composition of the invention.

The invention will now be illustrated by the following examples. Theexamples are intended to illustrate the invention but not limit itsscope.

EXAMPLES Example 1

Strain:—Thalassiosira sp. Thalassiosira sp. is a diatom, and the strainused was isolated from Bay of Bengal. This strain dominates duringsummer months, and it was isolated from seawater collected near Chennai,India. This culture was maintained in open tubs. The strain wasidentified as Thalassiosira weissflogii. This strain was capable ofgrowth at high temperatures (35-38° C.). The fatty acid profile was goodeven when the alga was grown at high temperature—with 25-30% EPA (aspercentage of fatty acids).

Culturing

The lab cultures were maintained in tubs in artificial seawater medium,under fluorescent lights (3000-4000 lux) and the temperature wasmaintained at 25° C.

Initial expansion of the culture was done under laboratory condition intubs. The dilution rate was 15% to 30% of the total culture volume perday. Once the volume was 40-50 liters, it was transferred to an outdoorpond. The outdoor ponds were covered with netting to control the light(40000 to 50000 lux). The dilution continued until the culture reached100,000 liters volume. The culture was held in 500 sq. m ponds at thistime, with a culture depth of 20 cm. The culture was stirred with apaddle wheel and CO₂ was mixed to keep the culture pH neutral. When theEPA levels in the pond reached a desirable level (10-15 mg/lit), thewhole pond was harvested by filtration. The filtered biomass was washedwith saltwater (15 parts per thousand concentration) and then spraydried. The mode of culturing was batch mode. The EPA productivity was2-3 mg/lit/day.

The ponds can also be run continuously for several weeks by harvestingpart of the culture, recycling the filtrate into the ponds andreplenishing required nutrients.

Example 2

Strain: Tetraselmis sp. Tetraselmis sp. is in the division Chlorophytaand the class Prosinophyceae or Micromanadophyceae. This strain wasobtained from the Central Marine Fisheries Research Institute, India. Itwas isolated from the local marine habitats in India. The culture wasmaintained in flasks in artificial seawater medium, and expanded asdescribed for Thalassiosira. With culture outdoors in open ponds asdescribed for Thalassiosira, the strain gave a good lipid yield (200-300mg/liter) and an EPA content of 6-7% of fatty acids.

Example 3

Strain: Chaetoceros sp. This is another diatom strain obtained from theCentral Marine Fisheries Research Institute, India, and isolated fromlocal marine habitats in India. Chaetoceros sp. was maintained in flasksand cultivated in outdoor ponds photoautotrophically as described inExample 1. It gave similar EPA productivity and EPA content asThalassiosira, described in Example 1.

Example 4

Strain: Isochrysis sp. Isochrysis is in the Prymnesiophyta, classPrymnesiophyceae, order Isochrysidales. It was obtained from the CentralMarine Fisheries Research Institute, India, and isolated from localmarine habitats in India. It was maintained and grown as described inExample 1. It was expanded from laboratory culture to a 50,000 literoutdoor pond culture in 14-15 days with a dilution rate of 15-30% perday. The lipid content at harvest was 100-150 mg lipids/liter. The rateof lipid production was 25-50 mg/liter/day. DHA was 10-12% of totalfatty acids.

Example 5

Harvesting and Drying:

The harvesting may be done by flocculation. The commonly usedflocculants include Alum with polymer; FeCl₃ with or without polymer andchitosan. The concentration of flocculent will depend on the cell numberin the culture before harvest. The range may vary from 100 ppm to 500ppm. Alternatively harvesting is done by filtration using appropriatemeshes. Removal of adhered chemicals (other than salt) is effected bywashing the cells in low salinity water.

The harvested slurry is then taken for spray drying. If required theslurry is sometimes encapsulated to prevent oxidation. The concentrationof encapsulating agent may vary from 0.1 to 1.0% on dry weight basis.Modified starch is a suitable encapsulating agent. The spray dryer usedis of atomizer or nozzle type. The inlet temperature ranges from 160 to190° C. and the outlet temperature ranges from 60 to 90° C. The spraydried powder is used immediately for extraction. If storage is needed,the powder is packed in aluminum laminated pouches and sealed afterdisplacing the air by nitrogen. The packed powder is stored at ambienttemperature until further use.

Example 6

Extraction:

Extraction of EPA/DHA is carried out using wet slurry or dry powder.Extraction is carried out using solvents. The solvents include hexane,ethanol, methanol, acetone, ethyl acetate, isopropanol and cyclohexaneand water. The above solvents are used alone or a combination of twosolvents. The solvent to biomass ratio depends on the starting material.If it is a slurry the ratio is 1:2 to 1:10. In the case of a spray driedpowder, the ratio is 1:4 to 1:30. The extraction is carried out in anextraction vessel under inert atmosphere. Extraction temperature rangesfrom 25 to 60° C. and the time varies from one hour to 10 hours. Solventaddition is made one time or in parts based on the lipid level in thecells. After extraction of crude lipid, the mixture is passed through acentrifuge or filtration system to remove the cell debris. The lipid inthe filtrate is then concentrated by removing the solvent bydistillation. The distillation process is carried out under vacuum. Theresulting product is a crude lipid extract, which contains approximately10% omega 3 fatty acid (EPA/DHA). This lipid extract can be used as suchor purified further to enrich the omega 3 fatty acids. Furtherpurification may involve removal of unsaponifiables such as pigments,sterols and their esters.

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What is claimed is:
 1. A concentrated microalgae composition prepared bya process comprising: (a) cultivating microalgae photoautotrophicallyoutdoors in open ponds; and (b) concentrating the microalgae, whereinthe microalgae are Thalassiosira sp., to obtain said microalgaecomposition containing at least 40% by weight of lipids in the form ofglycodiacylglycerides, phosphodiacylglycerides, or a combination thereofand at least 5% by weight of fatty acids selected from the groupconsisting of DHA, EPA, and a combination thereof.
 2. The concentratedmicroalgae composition of claim 1 wherein at least 20% by weight of thefatty acids in the composition are eicosapentaenoic acid (EPA).
 3. Theconcentrated microalgae composition of claim 1 wherein yield of EPA inthe cultivated microalgae is at least 10 mg/liter culture.
 4. Theconcentrated microalgae composition of claim 1, wherein the concentratedmicroalgae composition is prepared by a process further comprising:cultivating microalgae photoautotrophically outdoors in open ponds underfiltered sunlight in continuous or batch mode at a dilution rate of lessthan 35% per day; and harvesting the microalgae in exponential phasewhen cell number is increasing at a rate of at least 20% of maximalrate.
 5. The concentrated microalgae composition of claim 2 wherein themicroalgae are cultivated photoautotrophically outdoors in open pondsfor at least 14 days under filtered sunlight.
 6. The concentratedmicroalgae composition of claim 1 wherein the microalgae are cultivatedat above 20° C.
 7. The concentrated microalgae composition of claim 1wherein the microalgae are cultivated at above 30° C.
 8. Theconcentrated microalgae composition of claim 1 wherein the microalgaeare cultivated photoautotrophically outdoors in open ponds for at least14 days under filtered sunlight.
 9. The concentrated microalgaecomposition of claim 1 wherein the composition comprises anencapsulating agent to prevent lipid oxidation.
 10. An aquaculture oranimal feed comprising: the concentrated microalgae composition ofclaim
 1. 11. A purified lipid composition prepared from the concentratedmicroalgae composition of claim 1 by a process comprising: extractingthe lipids from the concentrated microalgae composition of claim 1 toobtain said purified lipids of the purified lipid composition comprisingat least 5% by weight EPA or DHA or a combination thereof.
 12. Thepurified lipid composition of claim 11 wherein at least 20% by weight ofthe fatty acids in the microalgae composition are eicosapentaenoic acid(EPA).
 13. The purified lipid composition of claim 11 wherein yield ofEPA in the cultivated microalgae in said step (a) is at least 10mg/liter culture.
 14. The purified lipid composition of claim 11,wherein the concentrated microalgae composition is prepared by a processfurther comprising: cultivating microalgae photoautotrophically outdoorsin open ponds under filtered sunlight in continuous or batch mode at adilution rate of less than 35% per day; and harvesting the microalgae inexponential phase when cell number is increasing at a rate of at least20% of maximal rate.
 15. The purified lipid composition of claim 12wherein the microalgae are cultivated photoautotrophically outdoors inopen ponds for at least 14 days under filtered sunlight.
 16. Thepurified lipid composition of claim 11 wherein the microalgae arecultivated at above 20° C.
 17. The purified lipid composition of claim11 wherein the microalgae are cultivated at above 30° C.
 18. Thepurified lipid composition of claim 11 wherein the microalgae arecultivated photoautotrophically outdoors in open ponds for at least 14days under filtered sunlight.